大肠杆菌HPr的活性位点组氨酸与信号转导蛋白IIIGlc之间磷酰基转移的速率常数和平衡常数。

Rate and equilibrium constants for phosphoryltransfer between active site histidines of Escherichia coli HPr and the signal transducing protein IIIGlc.

作者信息

Meadow N D, Roseman S

机构信息

Department of Biology and the McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland 21218, USA.

出版信息

J Biol Chem. 1996 Dec 27;271(52):33440-5. doi: 10.1074/jbc.271.52.33440.

DOI:10.1074/jbc.271.52.33440

PMID:8969207

Abstract

The bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS) plays a central role in catabolizing many sugars; regulation is effected by phosphorylation of PTS proteins. In Escherichia coli, the phosphoryltransfer sequence for glucose uptake is: PEP --> Enzyme I(His191) --> HPr(His15) --> IIIGlc(His90) --> IIGlc(Cys421) --> glucose. A rapid quench method has now been developed for determining the rate and equilibrium constants of these reactions. The method was validated by control experiments, and gave the following results for phosphoryltransfer between the following protein pairs. For phospho-HPr/IIIGlc (and HPr/phospho-IIIGlc), k1 = 6.1 x 10(7) M-1 s-1, k-1 = 4.7 x 10(7); for the mutant H75QIIIGlc in place of IIIGlc, k1 = 2.8 x 10(5) M-1 s-1, k-1 = 2.3 x 10(5). The derived Keq values agreed with the Keq obtained without use of the rapid quench apparatus. Keq for both reactions is 1-1.5. The rate of phosphoryltransfer between HPr and wild type IIIGlc is close to a diffusion-controlled process, while the reactions involving the mutant H75QIIIGlc are 200-fold slower. These rate differences are explained by an hypothesis for the mechanism of phosphoryltransfer between HPr and IIIGlc based on the structures of mutant and wild type proteins (see Pelton et al. (Pelton, J. G., Torchia, D. A., Remington, S. J., Murphy, K. P., Meadow, N. D., and Roseman, S. (1996) J. Biol. Chem. 271, 33446-33456)).

摘要

细菌磷酸烯醇丙酮酸

葡萄糖磷酸转移酶系统（PTS）在多种糖类的分解代谢中起核心作用；其调节通过PTS蛋白的磷酸化来实现。在大肠杆菌中，葡萄糖摄取的磷酸转移序列为：磷酸烯醇丙酮酸→酶I（组氨酸191）→HPr（组氨酸15）→IIIGlc（组氨酸90）→IIGlc（半胱氨酸421）→葡萄糖。现已开发出一种快速淬灭方法来测定这些反应的速率和平衡常数。该方法通过对照实验得到验证，并给出了以下蛋白质对之间磷酸转移的结果。对于磷酸 - HPr/IIIGlc（以及HPr/磷酸 - IIIGlc），k1 = 6.1×10⁷ M⁻¹ s⁻¹，k⁻¹ = 4.7×10⁷；用突变体H75QIIIGlc代替IIIGlc时，k1 = 2.8×10⁵ M⁻¹ s⁻¹，k⁻¹ = 2.3×10⁵。推导得到的平衡常数Keq值与不使用快速淬灭装置时得到的Keq值一致。两个反应的Keq均为1 - 1.5。HPr与野生型IIIGlc之间的磷酸转移速率接近扩散控制过程，而涉及突变体H75QIIIGlc的反应则慢200倍。基于突变体和野生型蛋白质的结构，对HPr和IIIGlc之间磷酸转移机制的一种假说解释了这些速率差异（见Pelton等人（Pelton, J. G., Torchia, D. A., Remington, S. J., Murphy, K. P., Meadow, N. D., and Roseman, S. (1996) J. Biol. Chem. 271, 33446 - 33456））。